Stable aerosol formulations of peptides and protein with non-cfc propellants

ABSTRACT

Glycosidicaily stabilised macromolecules, such as proteins and peptides, have substantially greater stability in the presence of hydrofluoroalkane propellants for dispensing from metered dose inhalers, when formulated with polyhydroxylated polyalkenes such as PVA.

The present invention relates to glycosidically stabilised preparationsof therapeutic materials for use in metered dose inhalation devices, andmethods for their preparation.

Pulmonary delivery has been employed for many years for drugs intendedto have localised, rather than systemic, effects. Essentially, there arethree types of device available for pulmonary delivery, and these arenebulisers, metered dose inhalers (MDI) and dry powder inhalers (DPI).Each of these has its benefits and its drawbacks.

Nebulisers are particularly effective for the administration of aqueousformulations of drug to non-ambulatory patients. Drug solution isconverted into microdroplets which are inhaled by the patient, thesemicrodroplets providing the facility to deliver the drug in a variety ofdose volumes, ranging from several milligrams to grams. However,nebulisers are generally large and unsuitable for ambulatory use, andthere is a problem with the potential instability of drugs in aqueoussolution, as well as during the process of nebulisation. In addition,reproducible dosing can be difficult with these devices.

MDI's are the most widely used pharmaceutical inhalation devices. Theformulations used in these devices routinely comprise drug, propellants,and stabilising excipients. In general, the drug is formulated togetherwith the excipients and then combined with the propellants, underpressure, to form either a suspension or solution formulation. Fine,respirable particles of drug are then produced as a consequence of thebreak up of droplets expelled from the device under pressure, followedby extremely rapid evaporation of the propellants. The amount of drug iscontrolled by delivering a pre-metered volume of propellant/drugmixture.

The suitability of MDI's to deliver peptide and protein pharmaceuticalshas not been well established, and there are concerns for the physicaland chemical stability of formulated proteins and peptide particles inpropellant mixtures. For these reasons, and the ability to deliver moresubstantial quantities, DPI's have been generally preferred for theinitial research into pulmonary delivery of proteins and peptides.

However, unlike MDI's, the ergonomics of DPI's are manufacture-dependentand, as a result, this can cause confusion amongst patients, which canlead to poor efficacy of therapy. In one study, 40% of patients who hadbeen taught how to use a Turbuhaler®, and who had used it for between 8months to 8 years, used it so poorly that it was unlikely that thepatients were obtaining any therapeutic benefit from the inhaled drugs.

In addition, where the amount of drug to be delivered is not an issue,then the benefits of using DPI's over MDI's is equivocal. In recentstudies, there was no evidence that DPI's were any more effective indelivering corticosteroids and β-2 agonist bronchodilators in asthmathan MDI's

Furthermore, the aerodynamic performance of MDI and DPI devicescontaining the same glucocorticoid was compared in vitro, and it wasestablished that the fine particle mass (FPM) delivered by the DPI wasflow rate dependent and significantly lower than that achieved using theMDI.

Thus, the primary advantage of DPI's lies in their ability to dispenselarge quantities of drug from a stable, powder formulation. By contrast,MDI's are able to dispense formulation in a more controlled, and moreeffective manner, but are more susceptible to physical instabilitychanges. A loss of physical stability can lead to particle aggregationand a lowering in the respirable fraction, or both.

MDI's are propellant-based delivery systems which, until recently,relied on the use of chlorofluorocarbons, or CFC's[trichlorofluoromethane (CFC-11) dichlorofluoromethane (CFC-12) and1,2-dichlorotetrafluoroethane (CFC-114)], in varying ratios, as theprincipal component of the formulation. With the universal, phasedwithdrawal of the use of CFC's, the only two propellants currentlyapproved for inhalation are tetrafluoroethane (HFA-134a) andheptafluoropropane (HFA-227). Both of these hydrofluoroalkanes haveboiling points substantially below 0° C., unlike CFC-11 (23.8° C.). Inaddition, the HFA's have poor solvency for those surfactants commonlyemployed as excipients in CFC-based MDI's, thereby further complicatingthe formulation design.

To date, the two most commonly employed formulation strategies for newBFA based MDI's include either the addition of a co-solvent, such asethanol, to generate a solution MDI, or the incorporation of novelstabilising excipients that are soluble in HFA's to form a suspensionMDI. Addition of a co-solvent to a drug-propellant mix can enhance thesolubility of the drug to a point where it is completely dissolved inthe BHA vehicle. As a consequence, a solution MDI generates respirableparticles in a different manner to more traditional suspensionformulations. Within a suspension MDI, particles of a defined size havealready been manufactured and simply require safe storage and deliveryby the device. However, a solution uses the design of the device and theenergy created by the evaporating solvent to form the particles uponactuation of the metering valve. The size of the particles ejected froma solution MDI is, therefore, heavily dependent on the actuation orificediameter and the device design (Lewis et al., 1998). Several researchgroups have demonstrated that optimisation of these two parameters canpotentially produce a dramatic increase in the delivery efficiency ofthe MDI compared to suspension based formulations (LeBelle et al.,1996;Stein, 1999).

There are, however, several fundamental flaws with formulating an MDI asa solution, including: lack of specific drug targeting; reduced chemicaland physical stability; and, a loss of control over the dissolution rate(Leach et al., 2002). The lack of control over the specific targetingwithin the deep lung has recently been studied by Hochhaus et al. Thisgroup described mean pulmonary residence time as the major influence onpulmonary targeting of steroids. They showed that solution MDI's had amuch lower pulmonary resonance time compared to suspension formulationsand suggested that this could result in a lack of lung steroid receptorspecificity, hence an increased chance of side effects (Hochhaus et al.,1998). However, by far the most difficult problem to overcome whenmanufacturing solution MDI's the reduction in the chemical stability ofthe drug (Sonie et al., 1992).

Blondino and Byron investigated the effects of a solution formulation onthe chemical stability of a model drug acetylsalicyclic acid (Blondinoand Byron, 1998). Results from this work indicated that inclusion of aco-solvent to enhance the drug-excipient-propellant compatibility alsoincreased the chemical degradation of the drug. In this study, this wasfound to be dependent on the concentration of surfactanti Furthermore,within a solution formulation, the drug is exposed to the significantlevels of dissolved water taken up in the HFA propellant (Vervaet andByron, 1999), and this can also induce chemical degradation.Manufacturing an MDI formulation as a solution tends, therefore, to losethe prime advantage of the dosage form, which should be to provide aprotective, apolar environment, which enhances both chemical andphysical stability.

A suspension based MDI overcomes the fundamental flaws associated withsolution formulations. A physically stable suspension of a therapeuticagent within a propellant provides a protective environment from whichparticles can be combined with numerous excipients to potentiallyachieve a versatile range of drug delivery properties. However, manytherapeutic agents require additional stabilising excipients to overcomethe problems associated with long-term physical stability within theformulation. The traditional excipients cannot be used for this purposedue to the switch of MDI propellants from CFC's to HFA's.

The formulation and delivery of macromolecules is substantially moredifficult than for the more commonly used low molecular weight organiccompounds. One of the major reasons for this is added complexity of thestructural make up of macromolecules. Proteins, for example, have up tofour levels of structural hierarchy including primary, secondary,tertiary and quaternary structures. If such compounds are to be used astherapeutic agents, they must be stored in a formulation and deliveredto the site of action with minimal changes to these structuralproperties, as failure to do so could result in reduction or completeloss of therapeutic activity, and may also lead to immunogenicity.

To date, recombinant human deoxyribonuclease I is the only therapeuticprotein specifically formulated for delivery to the lung. Recombinanthuman deoxyribonuclease is a hydrophilic glycosylated molecule with amolecular weight of ˜33 kDa. It is commercially available as Pulmozymegin the form of a nebuliser solution. It breaks down the viscosity oflung secretions of cystic fibrosis patients by digesting the endogenousDNA, which can be present at levels of up to 14 mg/ml in some cases.This digestion reduces the viscosity and facilitates the removal of themucus from the lung (Gonda, 1996). However, atomisation using anebuliser can deliver less than 30% of the drug to the lungs (Clarke etal., 1993), while the machine is bulky and difficult to use. Further,Pulmozyme® in solution is highly susceptible to heat degradation and hasto be stored below 8° C. and hence would not be considered an idealformulation.

The advantages of delivering proteins using MDI or DPI devices would besignificant, if the technological challenges can be overcome. It is ofprimary importance to maintain the stability of peptide and proteindrugs during processing and storage, as well as ensuring the efficiencyand reproducibility of the deposition of drug particles during use bythe patient. Particular considerations for MDI's include; the productionof particles with controlled particle size and stability, andcompatibility between propellants and the proteins and peptides. Suchfactors ensure that the suspension and biological stability can bemaintained over the required shelf life.

The stabilisation of proteins using compounds, such as sugars, hasenabled these complex macromolecules to be processed using a widevariety of manufacturing techniques with a minimal loss in therapeuticactivity (Allison et al., 1999;Aoudia and Zana, 1998a;Aoudia and Zana,1998b;Byron et al., 1996;Guiavarc'h et al., ;Imamura et al., 2003). Ofthe numerous processing methods used, spray-drying is one of the mostsuitable to produce inhalable particles, as the surface morphology ofthe particle can be manipulated Berggren, 2003; Chan et al., 1997;Harlow, 1993; Prinn et al., 2002; Stahl et al., 2002). However, proteinscannot commonly be spray-dried alone, as the heat used to dry theparticles denatures them, so that additional stabilising excipients arerequired to protect the molecule during the particulate manufacture.Although there have been many previous studies investigating the abilityof compounds to protect against the stresses induced during proteinmanipulation, little has been done to investigate the effects of suchexcipients on the performance of the final formulation. Although sugarscan protect against temperature-induced changes during processing, theydo little to protect against solvent-induced protein unfolding,hydrolysis, or aggregation-induced denaturation within a formulation.There is, therefore, a requirement to not only incorporate excipients toprotect the protein during manufacture into a suitable particle, butalso to maximise stability in the final delivery device.

The compatibility of BFA propellants with protein-powders has beeninvestigated in a number of previous studies. For example, Quinn et al.found that protein MDI formulations retained the biological activity oftested peptides and proteins, such as calcitonin and deoxyribonucleaseI, and found that the conformation of lysozyme underwent no change inthe presence of HFA-134a as analysed by Fourier transform Ramanspectroscopy (Quinn et al., 1999). In other studies, workers from 3MLimited found that protein MDI formulations retained the biologicalactivity of tested peptides and proteins, such as calcitonin anddeoxyribonuclease I. Other work also suggests that MDI proteinformulations might be efficient in terms of aerodynamic performance andreproducibility, in terms of dosimetry.

Accordingly, if it were possible to provide MDI formulations of proteinhaving both suitable chemical and physical stability during manufactureand storage, then MDI's would have substantial advantages over DPI's forthe delivery of appropriate therapeutic substances.

Surprisingly, we have now found that glycosidically stabilised complexdrugs, or macromolecules, such as proteins and peptides, havesubstantially greater stability in the presence of HFA's, whenformulated with polyhydroxylated polyalkenes, such as PVA.

Accordingly, in a first aspect, the present invention provides aformulation of a therapeutic substance suitable for delivery to apatient by a metered dose inhalation device, the formulation comprisinga substantially dry powder preparation of the substance in associationwith a stabilising amount of a glycoside and a polyhydroxylatedpolyalkene in combination with one or more propellants therefor.

In an alternative aspect, the present invention provides a formulationof a therapeutic substance suitable for delivery to a patient by ametered dose inhalation device, the substance being in association witha stabilising amount of a glycoside and being formulated in one or morepropellants and/or cosolvent, characterised in that the therapeuticsubstance is first prepared as a substantially dry powder in thepresence of a polyhydroxylated polyalkene, prior to formulation withpropellant.

Preferred therapeutic substances are peptides and proteins, andespecially those capable of having a therapeutic effect via oral ornasal administration from a metered dose inhaler. The protein or peptidemay act in situ, or systemically. A particularly preferred substance isdnase I, preferably human or humanised dnase I, especially dnase Isubstantially indistinguishable from naturally occurring human dnase Iin amino acid sequence or tertiary structure. Human dnase I is mostpreferred.

In particular, we have now found that dnase, for example, can beformulated with a polyhydroxylated polyalkene and a glycoside in an MDIto retain both biological activity and structural integrity during theproduction of respirable particles and formulating the particles withBFA propellant. Without these additives, there is a dramatic loss inactivity together with structural changes when dnase is spray-driedalone. Thus, it appears that the sugar and the polymer, in combination,protect the protein from both heat-induced denaturation duringspray-drying and solvent induced changes upon formulation. It is also anadvantage that the formulations of the invention are less likely to beimmunogenic, as the additives tend to stabilise the conformation of theactive molecule.

Where reference is had to dnase, herein, it will be appreciated thatsuch reference includes reference to all suitable therapeuticsubstances, unless otherwise apparent, or indicated.

It is a further advantage that the formulations of the invention can beused with portable MDI devices which are easy to use. In addition, thestabilisation of the protein allows it to be stored at room temperature.The delivery efficiency also tends to be higher than with nebulisers,while the delivered protein also generally has significantly greateractivity than in a nebulisable formulation.

Therapeutic substances are generally any substances suitable foradministration via an MDI device for therapeutic purposes, whether forprophylaxis or treatment. In general, therapeutic substances suitablefor use in the formulations of the present invention are advantageouslylarger, organic molecules, such as peptides and proteins, and mayinclude therapeutic glycosides and steroids, for example. Such moleculesmay have substantial stability in the presence of BFA's, but themajority of peptides and proteins are not conformationally stable overlong periods, and may lose activity, or physical stability, or oftenboth. This loss of activity arises not only through degeneration of thepeptide or protein, but also from aggregation of the suspendedformulation particles, which serves to reduce the fine particle masscritical for the treatment of the patient.

Such large organic molecules may be stabilised by the presence ofsuitable glycosidic compounds, particularly the lower oligosaccharides,particularly the di-, tar-, and tetra-saccharides. The terms“glycosides” and “glycosidic compounds” are used interchangeably herein.The composition of the oligosaccharide is not critical to the presentinvention, and the molecule may comprise a furanosyl residues, pyranosylresidues, straight chain elements, or mixtures thereof. For example,sucrose comprises a furanosyl and a pyranosyl residue, whilst mannitolcomprises a pyranosyl residue and a straight chain element. Othersuitable disaccharides include lactose, isomaltose, cellobiose, maltoseand trehalose, of which trehalose is preferred. Other suitableoligosaccharides include raffinose, melezitose and stachyose. It will beappreciated that the present invention envisages the use of any ofthese, or other, oligosaccharides either individually or as mixtures. Aparticularly preferred glycosidic compound is trehalose.

Other glycosidic compounds that may be used include such compounds asmannitol, xylitol, sorbitol, maltitol, isomalt and lactitol. Suitableamounts of the glycosidic compounds are, very approximately, on paritywith the therapeutic substance, by weight. More generally, the amount ofglycosidic compounds may vary between about 30% and 400% by weight ofthe therapeutic substance.

It will be appreciated that the glycosidic compounds are preferablysimply carbohydrate compounds, but the present invention also includesderivatives thereof, including the glucuronides.

It is an advantage of the present invention that, by combination with aglycoside and a suitably substituted polyhydroxylated polyalkene, thetherapeutic substances are now able to be provided in formulations whichare stable, even in the presence of haloalkane propellants. It is aparticular advantage that such stability is demonstrated in the presenceof HFA's, but it will be appreciated that such stability is alsodemonstrated in the presence of other propellants, such as CFC's, andalkanes, such as butane and propane or combinations of said propellants.

Preferred propellants are the haloalkanes, and it is preferablyenvisaged that BFA's are used as propellants for MDI's in formulationsof the present invention. However, it will be appreciated that theinvention also extends to the use of CPC's and other alkanes, forexample. The backbone of the propellant will generally be an alkane,whether substituted or unsubstituted, and may be straight or branched.Where branched, it is preferred that there only be one branch. Straightchains of the lower alkanes are preferred, especially C₂₋₄.

The preferred HFA's for use in the present invention are HFA-134a andHFA-227.

Suitable polyhydroxylated polyalkenes for use in the present inventionpreferably have the structure—(CH₂—CHOR)_(n)—where R is the same or different from one monomeric unit to the next,and is hydrogen, lower alkyl, lower alkenyl, lower alkanoyl, loweralenoyl or is a bridging group between adjacent monomers, such as alower diacyl group. By “lower” is meant 1 to 6 carbon atoms, other thanthe carbonyl carbon, where present, with 1 to 4 being more preferred,and 1 or 2 being more preferred.

Examples of suitable polyhydroxylated polyalkenes include PVA, PVAc(polyvinylalcohol and polyvinylacetate, respectively),- polyvinylalcohol-co-vinyl acetate (PVAA), poly(vinyl butyral) and poly(vinylalcohol-co-ethylene).

PVA is generally prepared by the hydrolysis of PVAc, and the level ofhydrolysis may be as low as about 40% through to substantially completehydrolysis, such as 98% or higher. High levels of hydrolysis correspondto lower levels of hydrophilicity/higher levels of hydrophobicity, whichcan affect the formulations of the present invention. It is generallypreferred that the level of hydrolysis be in the region of 50 to 90%,with a level of about 80% being a preferred embodiment.

The size of the polyhydroxylated polyalkene compounds is not critical tothe present invention, and PVA may range from a molecular weight of 9kDa through to about 500 kDa, with 9 kDa to 50 kDa being more preferred.Where PVA is used as the sole polyhydroxylated polyalkene, then apreferred molecular weight is in the region of 10 kDa. It will beappreciated that molecular weights for the polyhydroxylated polyalkenesare necessarily highly approximate, as the methods for their preparationnecessarily result in a spread of molecular sizes.

Suitable amounts of polyhydroxylated polyalkenes range from about 5% toabout 200% by weight of the therapeutic substance, although there islittle advantage to be seen in the provision of large amounts of thepolyhydroxylated polyalene. In general, a suitable amount ofpolyhydroxylated polyalkene is between about 10% and about 50% by weightof the therapeutic substance with a range of about 20% to about 40%being preferred.

Prior to formulation with the haloalkane propellant, it is preferred toblend the therapeutic agent with the glycosidic compound andpolyhydroxylated polyalkene in an aqueous vehicle, prior to drying. Theaqueous vehicle may be any suitable, and will typically be selected fromsaline or a suitable buffer such as phosphate buffered saline (PBS),although deionised water may also be used, if desired.

It will be appreciated that some formulations may comprise two or morepopulations of particles for administration. In such instances, theglycosides and polyhydroxylated polyalkenes may be selected asappropriate to each substance, and combined with propellant onceprepared. It is also possible that, where there are two or more activesubstances, any two or more may be formulated together.

The powdered products resulting from the drying of the aqueouspreparation may be achieved by any suitable drying process, includingfreeze-drying, spray-drying, spray-freeze-drying, supercritical drying,co-precipitation and air-drying. Of these, spray-drying andspray-freeze-drying are preferred, as these result in fine powders whichgenerally require no further processing. However, if required, the driedproducts may be further processed to reduce the size of the resultingparticles to an appropriate level. In particular, it is preferred thatthe aerodynamic diameter of the particles of the powder used in theformulations of the present invention is between about 1 μm and 50 μm,more particularly between about 1 μm and 12 μm, and even moreparticularly between about 1 μm and 10 μm. The dried powder is thenbrought into contact with the propellants under conditions suitable forstoring in a reservoir useful in an MDI.

It is a particular advantage of the present invention that the stabilityof the particles prepared as described above is considerably greaterthan anything provided in the art, and preferred formulations of thepresent invention comprise only the active ingredient(s), glycoside(s),polyhydroxylated polyalkene(s), and propellant(s). Thus, formulations ofthe present invention provide long-term stability of activity of thetherapeutic substance, as well as ensuring consistency of dosing withtime.

It will be appreciated that the present invention further provides apowdered formulation of a therapeutic agent, a glycoside and apolyhydroxylated polyalkene suitable for incorporation with a haloalkanepropellant for dispensing from a metered dose inhaler.

The present invention further provides a metered dose inhalation deviceprovided with a reservoir comprising a haloalkane propellant preparedwith a therapeutic substance, a glycoside and a polyhydroxylatedpolyalkene.

Doses delivered by the MDrs of the present invention will be readilydetermined by those skilled in the art and as appropriate to thecondition to be treated. In general, doses will vary with the size andage of the patient and can be readily determined by calculating theconcentration of the active ingredient in the propellant preparation.

Suitable macromolecular compounds for use as therapeutic agents includeantibodies, interferon, such as α-interferon, β-interferon andγ-interferon, enzymes such as proteases and ribonucleases, especiallyDNase L hormones, such as insulin, LHRH, granulocyte-colony stimulatingfactor, calcitonin, heparin, human growth hormone, euprolide acetate andparathyroid hormone and gene products such as CFTR, and α1-antitrypsin.Other large and/or complex molecules or structures may also beincorporated in MDI formulations, in accordance with the presentinvention.

The present invention will now be further illustrated by the following,non-limiting examples.

EXAMPLE 1

Compositions and Spray-Drying Process for Preparing Particles

Particles suitable for admixture with a propellant mixture were preparedas follows. Buffer phosphate salts (ACS reagent grade), sodium chloride,PVA (MW, 9,000-10,000), sucrose, trehalose, lysozyme, and catalase werepurchased from Sigma-Aldrich Co.

Enzymes and excipients were dissolved in buffer or saline andspray-dried using a Model 190 Butchi mini spray-dryer. The solutionsemployed to dissolve lysozyme and catalase were 5 mM sodium phosphatebuffer (pH 6.2) and 5 mM potassium phosphate buffer (H 7.0),respectively, and the enzyme concentrations were maintained at 5 mg/ml.The compositions of the spray-dried formulations are shown in Table 1,below. TABLE 1 The compositions and designations of spray-dried enzymeformulations. Formulation Composition LO1:0 Lysozyme 5 mg/ml LS1:1Lysozyme 5 mg/ml + sucrose 5 mg/ml LT1:1 Lysozyme 5 mg/ml + trehalose 5mg/ml LPT5:0.5:5.5 Lysozyme 5 mg/ml + PVA 0.5 mg/ml + trehalose 5.5mg/ml LPT5:1:6 Lysozyme 5 mg/ml + PVA 1 mg/ml + trehalose 6 mg/mlLPT5:2:7 Lysozyme 5 mg/ml + PVA 2 mg/ml + trehalose 7 mg/ml LPT1:1:2Lysozyme 5 mg/ml + PVA 5 mg/ml + trehalose 10 mg/ml CO1:0 Catalase 5mg/ml CS1:1 Catalase 5 mg/ml + sucrose 5 mg/ml CT1:1 Catalase 5 mg/ml +trehalose 5 mg/ml CPT5:1:6 Catalase 5 mg/ml + PVA 1 mg/ml + trehalose 6mg/ml

The feed solution was pumped peristaltically through a silicone tube (3mm) to a two fluid nozzle (0.5 mm) head used to atomise the fluid.Cooling water (0° C.) was circulated through the jacket around thenozzle at a-rate of about 36 ml/min. The processing parameters were: afeed rate of 3 ml/min; an atomising air-flow rate of 700 l/h; and aninlet temperature of 95° C. Outlet temperatures were found to range from65 to 69° C. The solution volume employed to produce each spray-dryingbatch was 100 ml and each process lasted 34 min. The protein powderswere collected in a collection jar, after all the feed solution had beenprocessed, but without allowing time for the powder to cool to roomtemperature, the material was transferred to a 7 ml vial, which wasimmediately sealed by capping. This vial was then transferred to afreezer (−20° C.) for storage.

EXAMPLE 2

Relative Enzyme Activity Remaining After Spray-Drying

The activity of the enzyme in each formulation is shown in Table 2.Spray-dried lysozyme was found to retain about 87% of the originalactivity, whilst those formulations containing excipients appeared tomaintain almost the full activity of the original enzyme. Inactivationof catalase upon spray-drying was found to be about 55% of the initialactivity, but the loss of activity was reduced to about 7% when eithersucrose or trehalose was included, and almost full activity wasrecovered when a PVA-trehalose mixture was included in the formulation.TABLE 2 Recovered biological activity in spray-dried lysozyme andcatalase particles (Mean ± SD, n = 3). Formulation Relative activity (%)Formulation Relative activity (%) LO1:0 87.2 ± 2.1 LPT1:1:2 95.9 ± 3.0LS1:1 97.2 ± 3.0 CO1:0 54.4 ± 4.1 LT1:1 96.9 ± 3.2 CS1:1 92.3 ± 2.8LPT5:0.5:5.5 100.4 ± 2.2  CT1:1 93.38 ± 2.1  LPT5:1:6 97.1 ± 3.7CPT5:1:6 99.8 ± 2.8 LPT5:2:7 97.3 ± 4.8

EXAMPLE3

Geometric Particle Size of Spray-Dried Particles

The particle size as well as size distribution of the spray-driedprotein particles are shown in Table 3. The volume median diameters(VMD) of all spray-dried particles were found to be between 2.48 and3.43 μm. In addition, the span of particle size distribution was foundto be between 0.77 and 1.18, which indicates that all the powdersexhibited a relatively high degree of monodispersity, whilst the upperlimit of the size range of the particles appeared to be ≦12.5 μm. TABLE3 Particle size and distribution of spray-dried lysozyme and catalaseformulations. Median diameter Size range Formulation (μm) Span (μm)LO1:0 3.09 0.77 1.22-7.49 LS1:1 3.43 0.92 1.22-12.5 LT1:1 3.31 0.911.22-11.6 LPT5:0.5:5.5 2.48 1.15 1.22-7.49 LPT5:1:6 2.67 1.03 1.22-9.31LPT5:2:7 2.78 1.18 1.22-10.0 LPT1:1:2 2.89 1.13 1.22-10.8 CO1:0 3.121.18 1.22-10.0 CS1:1 2.95 1.04 1.22-9.31 CT1:1 2.96 1.15 1.22-10.0CPT5:1:6 2.77 1.33 1.22-10.0

EXAMPLE 4

The Effect of HFA on the Biological Activity of Spray-Dried Enzymes

For spray-dried lysozyme and catalase in the presence of sucrose,trehalose or a trehalose-PVA mixture, there appeared to be no detectablereduction in activity after being stored in a BFA based-MDI canister upto 26 weeks, whilst the activity of spray-dried catalase alone was foundto be reduced to ˜20% within 12 weeks (Table 4). TABLE 4 The retainedactivity of HFA based MDI-formulated lysozyme and catalase particlesrelative to the corresponding control powders after storage for 12 weeksat room temperature. Activity of MDI- Activity of MDI-formulatedformulated enzyme at Formulation enzyme at Week-1 (%) Week-12 (%) LO1:099.0 98.1 LPT5:0.5:5.5 96.8 100.0  LPT5:1:6 102.9  95.3 LPT5:2:7 98.999.0 LPT1:1:2 97.6 93.0 CO1:0  98.9 ± 2.7 20.8 ± 6.8 CPT5:1:6 101.1 ±4.7 98.2 ± 1.8

EXAMPLE5

Deposition of MDI-Formulated Lysozyme Particles

The in vitro deposition performance of MDI-formulated spray-driedlysozyme particles is shown in Table 5. For the formulation preparedusing spray-dried lysozyme alone, the protein fractions recovered fromthe device, stage 1 and stage 2 were found to be about 14.6, 34.9 and50.5% respectively, during the first week after preparation. Afterstorage at room temperature for up to 12 weeks, the stage 2 fractionsignificantly decreased to 42.7% whilst the stage 1 fraction increasedto 42% of the recovered dose (p<0.05, one tailed student t-test, Table5).

When lysozyme was stabilised using either sucrose or trehalose asexcipient during spray-drying, the aerodynamic properties of theresultant MDI formulations were significantly affected (p<0.05, twotailed student t-test). At the first week after manufacture, the stage 2fraction of MDI formulation LS 1:1 appeared to decrease to 27.2% whilstthe fraction recovered from the device and stage 1 increased to 21.7 and51.3% respectively. After 6 weeks storage at room temperature, the stage2 fraction decreased significantly to about 8% (p<0.05, two tailedstudent t-test). However, with further storage for up to 26 weeks, thereappeared to be no more reduction in the stage 2 fraction. The MDIformulated LT1:1 particles displayed a similar aerodynamic performanceto the LS1:1 formulations at the first week after preparation. However,the storage suspension stability of the former proved to besignificantly better than the latter (p<0.05, paired student t-test).Nonetheless, the fine particle fraction (stage 2 fraction) of LT1:1 MDIformulation was susceptible to decrease as a function of storage time.After stored for 26 weeks, the fine particle fraction significantlydecreased to 12.7% (p<0.05, two tailed student t-test) whilst the stage1 fraction increased to 61.4% of the recovered protein.

When lysozyme was spray-dried in the presence of PVA-trehalose mixture,the resultant MDI formulations appeared to have significantly betteraerosol performance than those formulated using either trehalose orsucrose alone in combination with the enzyme (p<0.05, two tailed studentt-test, Table 5). The fine particle fraction of MDI formulationscontaining PVA were found to range from 47.1 to 52.7% at the first weekafter preparation. The stability of aerodynamic properties was found todepend upon the PVA content in the spray-dried particles. After storagefor 12 weeks at room temperature, the formulation (LPT5:0.5:5.5)containing the lowest PVA content was found to emit a insignificantlydecreased fine particle fraction of 42.8%, in comparison to the 48.3%obtained during week-1 (P>0.05, one tailed student t-test). The otherformulations containing a higher ratio of PVA content in theformulations appeared to retain a constant fine particle fraction overthe 12 week storage period. All the PVA containing MDI formulationsdisplayed a significantly better storage suspension stability, in termsof fine particle fraction, than either the MDI LS1:1 or LT1:1formulations p<0.05, paired student t-test). TABLE 5 The aerosolperformance of HFA based MDI lysozyme formulations as evaluated by atwin stage impinger after storage up to 12 weeks (Mean ± SD, n = 3).Fraction at Fraction at week- Fraction at Formulation Stage week-1 (%) 6(%) week-12 (%) LO1:0 Device 14.59 ± 3.21 15.41 ± 2.55 15.29 ± 3.11Stage 1 34.90 ± 2.75 34.27 ± 1.85 42.01 ± 2.89 Stage 2 50.51 ± 3.7650.32 ± 0.81 42.69 ± 3.41 LPT5:0.5:5.5 Device 15.97 ± 2.9  10.92 ± 1.0515.12 ± 3.56 Stage 1 37.54 ± 3.14 40.88 ± 2.73 42.07 ± 2.51 Stage 248.27 ± 3.3  48.20 ± 3.65 42.80 ± 3.89 LPT5:1:6 Device 15.20 ± 1.1015.30 ± 2.06 16.41 ± 2.78 Stage 1 39.33 ± 6.08 39.39 ± 1.97 38.72 ± 4.10Stage 2 47.14 ± 9.12 45.30 ± 0.30 44.87 ± 3.67 LPT5:2:7 Device 18.29 ±2.23 10.87 ± 1.03 13.49 ± 1.32 Stage 1 33.18 ± 6.60 41.65 ± 1.76 37.92 ±2.87 Stage 2 48.54 ± 4.46 49.11 ± 3.25 48.59 ± 3.2  LPT1:1:2 Device19.32 ± 2.97 13.35 ± 1.70 12.47 ± 3.21 Stage 1 30.57 ± 1.49 33.30 ± 4.3832.10 ± 2.98 Stage 2 52.73 ± 1.97 54.42 ± 3.10 55.44 ± 3.70

EXAMPLE 6

Deposition of MI-Formulated Catalase Particles

The in vitro deposition performance of MDI catalase formulations isshown in Table 6. For the MDI-formulated spray-dried catalase withoutany excipient, the protein fractions recovered form device, stage 1 andstage 2 were found to be about 23.7, 43.3 and 33.0% respectively, duringthe first week after preparation. However, after the same formulationshad been stored for 6 weeks at room temperature, the stage 2 fractionwas found to decrease drastically to almost 0% with about 89% ofparticles being deposited in stage 1 (Table 6). TABLE 6 The aerosolperformance of HFA based MDI-formulated catalase particles as evaluatedby a twin stage impinger after storage up to 12 weeks. Fraction atFraction at week- Fraction at Formulation Stage week-1 (%) 6 (%) week-12(%) CO1:0 Device 23.71 ± 2.09 10.00 ± 1.02 ND Stage 1 43.31 ± 1.32 88.79± 1.29 ND Stage 2 32.98 ± 0.90  1.20 ± 2.07 ND CPT5:1:6 Device 15.84 ±3.01 13.16 ± 2.58 17.85 ± 1.31 Stage 1 25.24 ± 1.00 32.47 ± 1.46 30.74 ±4.77 Stage 2 58.92 ± 3.02 54.37 ± 3.89 53.26 ± 6.08

The spray-dried catalase formulation containing either sucrose ortrehalose as stabiliser, produced a significantly higher stage 2deposition of protein relative to the MDI CO1:0 formulation, asevaluated during the first week after manufacture (p<0.05, two tailedstudent t-test). The stage 2 fractions of MDI formulated CS1:1 and CT1:1appeared to increase from 33.0% in the absence of excipient to 39.3 and44.8% respectively, when sucrose or trehalose were employed. The stage 1fractions appeared to be almost identical in the absence or presence ofexcipient. The fine particle fractions generated by the CS1:1 and CT1:1MDI formulations appeared to decrease as a function of storage time. Theformulation incorporating trehalose emitted a higher fine particlefraction after 6-26 weeks of storage than the similar formulationcontaining sucrose. For example, after 26 weeks storage at roomtemperature, the stage 2 fraction of the CS1:1 MDI formulation was 6.0%,relative to the 18.7% emitted from MDI containing the CT1:1 formulation.The reductions in the fine particle fractions were compensated byincreases in the stage 1 fractions, whilst the device fractions wereconsistently found to be about 20% of the recovered dose and independentof formulation and storage time.

The fine particle fraction of the PVA containing MDI formulation wasfound to be 58.9%, which was significantly higher than that of the MDIformulated CS1:1 or CT1:1 particles (p<0.05, two tailed student t-test),whilst the device and stage 1 fractions accounted for only 15.9 and25.2% of the recovered does respectively, as evaluated during the firstweek after preparation. After storage for 6 weeks at room temperature, aslight decrease in fine particle fraction was found, albeit notsignificant (p>0.05, one tailed student's t-test). Moreover, afterstorage for a further 6 weeks, the recovered fine particle fractionappeared to be the same. When catalase was spray-dried in the presenceof PVA-trehalose mixture, the resultant MDI formulations appeared alwaysto display a significantly better aerosol performance in comparison tothe MDI formulated CS1:1 or CT1:1 particles during storage (p<0.05,paired student t-test).

EXAMPLE 7

Stabilisation of a Dnase I Metered Dose Formulation

While the human form of deoxyribonuclease (dnase) is used for clinicalapplications, its manufacture and purification is costly. However, thebovine form of the protein provides an excellent model. The sequences ofthe human and bovine forms are 77% homologous and the crystal structurescan be superimposed upon each other (Quan et al., 1999). In thefollowing Example, highly purified bovine deoxyribonuclease I wasreformulated in a metered dose inhaler preparation, and the ability oftrehalose and polyvinyl alcohol to stabilise bovine dnase I duringmanufacture using spray-drying and formulation in a metered dose inhalerwas assessed, by comparison with spray-drying the raw enzyme alone.

Deoxyribonuclease I (isolated from the bovine pancreas, high purity,Rnase free, 14200 U/mg (defined by Sigma Aldrich as Genotech® units)Sigma Aldrich, Gillingham, UK) formulations were manufactured using theBucchi 191 mini spray-dryer (B3ucchi, Darmstadt, Germany). Theaspiration rate was set as 70%, the material feed rate was 3 ml min⁻¹and the inlet temperature was set to 95° C. The feed suspension waspumped through a spray atomisation nozzle that combined the liquid witha 700 ml hr⁻¹ airflow. The outlet temperature was determined by thepreviously detailed parameters but was consistently found to be in therange 65-70° C.

The dnase spray-drying feed solutions were made up in 100 ml of 0.15MNaCl buffer. Two formulations were manufactured in total as detailed inTable 7, below. The PVA was 80% hydrolysed with a molecular weight (M,)of 8,000-10,000 (Sigma Aldrich, Gillingham, UK). The trehalose was inthe dihydrate form (Sigma Aldrich, Gillingham, UK). TABLE 7 Compositionof the Deoxyribonuclease I spray-dried formulations FormulationComposition DO1:0 dnase I 5 mg/ml DTPVA 1:1:1 dnase I 5 mg/ml +trehalose 5 mg/ml + PVA 80% hydrolysed 5 mg/ml

The product from the spray-drying process was collected and weighed intoa glass vial. The samples were stored under phosphorous pentoxidedesiccation at room temperature for 24 hours prior to MDI manufacture.TABLE 8 Composition of the Deoxyribonuclease I MDI formulationsFormulation Composition DO1:0 134a DO1:0 15.0 mg + HFA 134a 15.0 g DTPVA1:1:1 DTPVA 1:1:1 45.0 mg + HFA 134a 15.0 g 134 a DTPVA 1:1:1 DTPVA1:1:1 45.0 mg + HFA 227 17.0 g 227

The metered dose inhalers were manufactured by adding the equivalent of15.0 mg of the raw drug (dnase) into a PET canister (BesPack, KingsLynn, UK), so that 15.0 mg of DO1:0 and 45.0 mg of DTPVA 1:1:1 wereused. A total of three formulations were manufactured, as detailed inTable 8, above. A 25 μL canister valve (BesPack, Kings Lynn, UK) wascrimped in place using the Pamasol MDI filler (Pamasol, Pfaffikon,Switzerland) and 15.0 g of HFA 134a (Dupont, Willington, Germany) or17.0 g HFA 227 (Solvay, Frankfurt, Germany) was pressure-filled into thecan via the valve. The formulation was then sonicated in anultrasonication bath (Decon, Hove, UK) for 15 seconds to ensure particleseparation and stored, valve up, at room temperature. The denatureddnase used as a positive control was simply manufactured by placing 5.0mg of the protein in a 180° C. oven for 10 minutes.

Particle Size Analysis

The spray-dried powders were assessed using the Mastersizer X laserdiffraction particle size analyser (Malvem Instruments Ltd, Malvem, UK).The Malvem was set up using the liquid dispersion system. Mixtures of 1%lecithin (Sigma Aldrich, Gillingham, UK) and cyclohexane (Merck, Poole,UK) were used as the dispersion media. Samples were prepared bysonicating 2 mg of powder in 2 ml of the dispersion media for 30seconds. The particle size was measured using the 63 mm (0.5-110 μm)lens set at a focal length of 145 mm, whilst stirring the cell on 75% offull power. The samples were added dropwise in to the stirred cell untilthe desired obscuration was achieved. Each sample was measured intriplicate and 3 batches from each sample were analysed.

Biological Activity

The biological activity of dnase I was monitored by assessing theenzyme's ability to digest the substrate, DNA. The substrate was made upin an acetate buffer (0.1 M, pH 5.0), containing 5 MM Mg²+. This wasprepared by dissolving 1.165 g of anhydrous sodium acetate (BDH, Mercklabs, Darmstadt, Germany), 0.355 g of acetic acid (Sigma Aldrich,Gillingham, UK), and 0.203 g of MgCl₂.6H₂O (Sigma Aldrich, Gillingham,UK), in 150 ml of purified water. 2 mg of fibrous DNA isolated from acalf thymus (Sigma Aldrich, Gillingham, UK) was dissolved in 52 ml ofthe acetate buffer by gently shaking overnight. The absorbance of thissubstrate solution at 260 nm was determined to be between 0.630 and0.690.

Prior to assessing the test samples, a dnase I standard, 2,000 Kunitzunits mg⁻¹ (Sigma Aldrich, Gillingham, UK), was used as a calibrant forthe activity assay. This standard was reconstituted by dissolving it in1.0 ml of 0.15 M NaCl solution. The solution was further diluted toobtain five separate standard solutions within the concentration rangeof 20-80 units ml⁻¹. All dilutions were performed using 0.15 M NaClsolution.

A lambda 5 UV spectrophotometer (Perkin-Elmer, Beaconsfield, UK) wasadjusted to a wavelength of 260 nm and 2.5 ml of substrate was placedinto a cuvette (10 mm light path) and incubated in a thermostatic cell(25° C.) for 3-4 minutes to allow temperature equilibration. Then, 0.5ml of diluted standard, or sample, was added and the solutions wereimmediately mixed by inversion. The increase in A₂₆₀ (ΔA₂₆₀) minutes wasrecorded as a function of time for 10-12 minutes. An activitycalibration curve was constructed by plotting the maximum ΔA₂₆₀ vs.Kunitz units mg⁻¹ of the standard dnase I vials. The dnase samples werediluted to attain a ΔA₂₆₀ within the calibration range and, hence,measure the equivalent Kunitz units. The Pierce Protein Assay® was thenused to quantify the protein, thereby to obtain the activity per mg.This was compared to the lyophilised raw dnase I to produce the %relative activity.

Twin-Stage Impinger

The twin stage impinger (Radleys, Saffron, UK) was set up as per theUnited States Pharmacopoeia specification. The dnase I formulations useddistilled water as washing agent and the solvent in the apparatus. Theairflow was set to 60 ml min⁻¹ and the inhalers were actuated 20 times.Between each actuation there was a five second pause with the pumprunning. The pump was then stopped, the canister removed and shaken forfive seconds before the sequence repeated. Each of the stages werewashed individually upon completion of the 20 canister actuations. Thedevice was washed into a 50 ml volumetric with stages 1 and 2 beingwashed into 100 ml volumetric flasks. The resulting solutions wereanalysed using the Pierce Protein Assay® (Pierce Chemical Company, UK).All twin stage runs were completed in triplicate.

The Pierce Protein Assay® was performed as per the manufacturer'sinstructions. BSA was used as the protein standard and a set of BSAsolutions between 2 and 20 μg were prepared by diluting the 2.0 mg ml⁻¹standard. The working reagent was prepared by mixing 25 parts of MicroBCA reagent A and 24 parts of reagent B with 1 part of reagent C. Analiquot of 150 μL of each standard or test sample was transferred into a96-well microplate in duplicate. 150 μL of the working reagent wassubsequently added to each well and the plate mixed on the shaker for 30seconds. The plate was covered and incubated at 50° C. for 90 minutes,after which it was cooled to room temperature and the UV absorbance ineach well determined at 562 nm using a UV plate reader. The response ofeach enzyme was determined by comparing the nominal concentration andthe BSA protein standard.

Fluorescence

Fluorescence emission and Rayleigh light scattering were both assessedusing a LS-50 fluorescence spectrophotometer with a thermostatic cellset at 5° C. (Perkin-Elmer, Beaconsfield, UK). The excitation wavelengthwas set to 270 nm and the emission was monitored over a range of 250 nmto 450 nm. The excitation slit width was set as 4 nm and the emissionslit width 8 mn. The spectra were attained at a rate of 150 nm. All thesamples were made up in a 0.15 M NaCl solution (Sigma Aldrich,Gillingham, UK). The samples were each scanned five times and averaged.The spectra from the solvent were subtracted from each result. The areaunder the light scattering peak (maximum cc. 270 nm) and thefluorescence peak (maximum cc. 335 nm) were integrated from each sampleand compared. The light source variance was assessed and, ifappropriate, corrected for, using Nile Red (Sigma Aldrich, Gillingham,UK) as a standard.

Spray-Dried Material Characterisation

The two formulations were manufactured using the Bucchi spray-dryer. Theparticle size measurements of the spray-dried material indicated thatboth of the batches were of a suitable respirable size, i.e. less than10 μm. The results are shown in Table 9. The smallest mean particle size(2.25±0.05 μm) was produced by simply spray-drying the protein alone.TABLE 9 Dnase I spray-dryer manufacture yield and particle sizedistribution (Mean ± SD, n = 3). D[v, 0.1] D[v, 0.5] D[v, 0.9]Formulation μm μm μm % Yield DO1:0 1.00 ± 0.02 2.25 ± 0.05 4.52 ± 0.5215.40 DTPVA 1:1:1 1.24 ± 0.01 3.06 ± 0.12 6.57 ± 0.43 34.75

The yields for the operation were improved with the addition of theexcipients. Incorporation of trehalose and PVA more than doubled theyield of the manufacture method from 15.40% to 34.75%.

The Effect of HFA on the Dnase I Formulations

The biological activity of the enzyme, dnase I, was reduced by almost40% when spray-dried alone from a 0.15 M NaCl solution. However, over85% of the biological activity was retained when PVA and trehalose wereincorporated into the feed solution. Results are shown in column 2 ofTable 10, below. The additional excipients showed a significantimprovement (p<0.05) in the retention of the enzyme's activity comparedto the enzyme spray-dried alone. TABLE 10 Relative biological activityof the spray-dried dnase particles before and after formulation in HFApropellants (Mean ± SD, n = 3). Dnase Particles HFA Formulation RelativeRelative Formulation activity (%) Formulation activity (%) DO1:0 63.86%± 4.23 DO1:0 134a 61.43% ± 3.88 DTPVA 1:1:1 85.34% ± 2.18 DTPVA 1:1:1134a  95.91% ± 10.98 DTPVA 1:1:1 227 103.06% ± 4.50 

Formulation of the DO1:1dnase I particles with HFA 134a had nosignificant effect on the activity of the enzyme, as shown in column 4of Table 10. However, surprisingly, addition of the DTPVA 1:1:1particles to both BFA 134a and HFA 227 reversed the 15% lost activity toreturn the enzyme to its original potency. There was no significantdifference between the activity of the dnase (p<0.05) in either of theHFA propellants. These results suggest that the when the protein isspray-dried alone, denaturation is occurring, but that this is preventedby combination with the excipients. The addition of PVA and trehaloseresults in total retention of the protein's activity, suggesting that,within the DTPVA 1:1:1 formulation, the active site of the protein, i.e. the area of the protein that digests the DNA, has remained intact.

In vitro prediction of particle deposition using the twin stage impingerapparatus defines the fine particle fraction as the particles collectedon stage 2 of the device. Stage 2 has a size cut off MMAD of <6.4 μm. Inaccompanying FIG. 1, there is presented the impaction data for the threednase MDI formulations, determined in vitro using a twin stage impinger.Formulations are detailed in Table 9.

Both sets of the spray-dried dnase particles (DO1:0 and the DTPVA 1:1:1)produced a high FPF in the twin stage impinger apparatus when suspendedin HFA propellants. DO1:0 134a delivered a significantly higher (p<0.05)FPF compared to either the DTPVA 1:1:1 134a or the DTPVA 1:1:1 227formulation (which were not significantly different (p<0.05) from eachother), see FIG. 1. However, the twin stage impinger data should not beanalysed in isolation. Both the percentage of dnase delivered to thesecond stage of the device and the activity of the particles deliveredmust be taken into account to attain a true prediction of formulationefficiency (FIG. 2). The accompanying FIG. 2 shows the combination ofenzyme activity data and twin stage impinger data to predict thequantity of active enzyme delivered to the lung.

Although the DO1:0 formulation delivers a high proportion of particlesto stage two, only 60% of these retained biological activity. The DTPVA1:1:1 formulation, on the other hand, retained fall biological activityand, therefore, when suspended in HFA, delivered more active particlesto stage 2. There was no difference between the two types of HFApropellants in this case, hence DTPVA 1:1:1 suspended in either HFA 134aor HFA 227 is the most efficient delivery vehicle for the protein. Bothformulations deposited almost 50% of the actuated dose on stage 2 of theTSI device.

Rayleigh light scattering is measured at 90 degrees to the incidentlight. The Rayleigh emission from particulates within solutions occursat the same wavelength at which it was applied to a sample. As moreaggregates are formed within a solution, the intensity of the Rayleighlight scattering increases. Therefore, measurement of Rayleigh lightemission has been previously used to monitor the aggregation of proteinsolutions. Aggregation follows secondary structure breakdown in aprotein and, therefore, may be indicative of protein denaturation.Furthermore, the tryptophan residue in a protein is known to befluorescent. Although this is not a unique property of amino acids (bothtyrosine and phenylalanine also fluoresce) the fluorescence of thetryptophan residue is uniquely sensitive to its microenvironment.Structural changes in a protein, such as unfolding or aggregation, canlead to change in the microenvironment of the tryptophan residue, whichresults in a change in fluorescent intensity, due to quenching orintensity maxima, through a variation in hydrophobicity of themicroenvironment. Hence, monitoring of Rayleigh light scattering (whichcan be performed in a single scan on a fluorescence spectrophotometer)and fluorescence can both indicate structural changes on both a macroand-Micro-environmental level. TABLE 11 Integrated light scattering andfluorescence peaks for the dnase I spray-dried material. Raleigh PeakFluorescence Peak Fluorescence Formulation Area Area Maximum DO1:0 73529± 4898 1546667 ± 202072 334.5 ± 0.5 DTPVA 1:1:1  1241 ± 2149 916143 ±18726 336.5 ± 0.5 DO1:0 134a 74036 ± 1530 1165807 ± 24109    333 ± 0.5DTPVA 1:1:1 134 a 24516 ± 247  684487 ± 7210  337 ± 0 DTPVA 1:1:1 22717647 ± 353  903542 ± 18070 336 ± 0

Assessing the particles prior to suspension in HFA propellanthighlighted the fact that the DTPVA 1:1:1 spray-dried formulationproduced a significantly lower (p<0.05) Rayleigh light scattering peakarea, compared to the DO1:0 particles, implying less protein aggregateswere present (see Table 11, above). However, the spray-dried dnase,DO1:0, produced a significantly (p<0.05) larger peak area for thefluorescence emission of tryptophan, compared to the DTPVA 1:1:1spray-dried formulation. Although the fluorescence maxima weresignificantly different (p<0.05) for the two dnase batches, thedifferences were so small that they were not considered indicative ofchanges in the protein structure. Upon suspension of the dnase particlesin HFA, changes were observed with both the Rayleigh light scatteringand the fluorescence emission spectra (again, the small shifts in peakmaxima, although statistically significant, were not considered, due tothe fact the shifts were so small they were thought not to be indicativeof structural changes). DO1:0 134 showed no significant change inRayleigh light scattering. However, it did show a significant drop(p<0.05) in fluorescence emission from a peak area of 1546667 to a peakarea of 1165807. DTPVA 1:1:1 134a also showed a drop in fluorescenceintensity after formulation in propellant, this was coupled with asignificant rise p<0.05) in Rayleigh light scattering. DTPVA 1:1:1 227observed a similar increase in Rayleigh light scattering to DTPVA 1:1:1134a, however, the fluorescence intensity remained constant both beforeand after incorporation with BFA propellant.

The changes in both Rayleigh light scattering and fluorescence intensityinfer that both the DO1:0 and the DTPVA 1:1:1 spray-dried particleschange upon suspension in BFA propellant. Only minimal changes occurredwith the DTPVA 1:1:1 227 formulation, indicative of enhanced stabilityfor the formulation.

Compared to a simple spraydried dnase formulation, incorporation of thetrehalose and PVA with the protein increased the yield of themanufacture method, improved the retention of the protein's activity,both before and after suspension in HFA, and maximised the secondarystructure integrity throughout. The PVA/ trehalose formulation alsominimised aggregate formation and slowed or prevented changes in themicroenvironment of the tryptophan residue.

The protection of the protein during both manufacture and theformulation of dnase I, using trehalose and PVA as excipients, produceda MDI formulation that was both aerodynamically suitable for lungdelivery and therapeutically active. The formulations held the proteinwithin its native state from the point of manufacture to its deliveryfrom the MDI device, which maximised its stability and minimises anypotential immunological responses by the body when it is delivered invivo.

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1. A formulation of a therapeutic substance suitable for delivery to apatient by a metered dose inhalation device, the formulation comprisinga substantially dry powder preparation of the substance in associationwith a stabilising amount of a carbohydrate compound or derivativethereof and a polyhydroxylated polyalkene in combination with one ormore propellants therefor, wherein the therapeutic substance is selectedfrom peptides and proteins.
 2. A formulation according to claim 1,further comprising a cosolvent for said substance.
 3. A formulationaccording to claim 1, wherein the therapeutic substance is selected fromantibodies, interferons, enzymes, hormones, euprolide acetate, CFTR, andα1-antitrypsin.
 4. A formulation according to claim 3, wherein thetherapeutic substance is a hormone selected from insulin, LHRH,granulocyte-colony stimulating factor, calcitonin, heparin, human growthhormone, and parathyroid hormone.
 5. A formulation according to claim 1,wherein the substance is dnase I.
 6. A formulation according to claim 1,which is non-immunogenic.
 7. A formulation according to claim 1, whichis capable of being stored at room temperature without losing more than50% biological activity of the therapeutic substance after two months.8. A formulation according to claim 1, wherein the carbohydrate compoundcomprises at least one oligosaccharide.
 9. A formulation according toclaim 8, wherein the carbohydrate compound comprises at least onedisaccharide.
 10. A formulation according to claim 9, wherein thedisaccharide is selected from trehalose, mannitol, sucrose, and mixturesthereof.
 11. A formulation according to claim 1, wherein thecarbohydrate compound or derivative thereof constitutes between about30% and 400% by weight of the therapeutic substance.
 12. A formulationaccording to claim 1, wherein the propellant is alkane based.
 13. Aformulation according to claim 12, wherein the propellant is at leastone haloalkane.
 14. A formulation according to claim 13, wherein thepropellant is selected from HFA-134a and HFA-227.
 15. A formulationaccording to claim 1, wherein at least one polyhydroxylated polyalkenehas the general structure —(CH₂—CHOR)_(n)— where R is the same ordifferent from one monomeric unit to the next, and is hydrogen, loweralkyl, lower alkenyl, lower alkanoyl, lower alenoyl or is a bridginggroup between adjacent monomers.
 16. A formulation according to claim15, wherein, when R is not hydrogen, the number of carbon atoms,excluding any —CO— group, is between 1 and 6, inclusive.
 17. Aformulation according to claim 15, wherein the polyhydroxylatedpolyalkene is selected from polyvinylalcohol, polyvinylacetate,polyvinyl alcohol-co-vinyl acetate, poly(vinyl butyral), poly(vinylalcohol-co-ethylene), and mixtures thereof.
 18. A formulation accordingto claim 17, wherein the polyhydroxylated polyalkene is PVA.
 19. Aformulation according to claim 18, wherein the PVA a hydrolysate ofPVAc, the level of hydrolysis being between 40% and 100%.
 20. Aformulation according to claim 18, wherein the PVA a hydrolysate ofPVAc, the level of hydrolysis being between 50 and 90%.
 21. Aformulation according to claim 18, wherein the PVA has a molecularweight of between about 9 kDa and 50 kDa.
 22. A formulation according toclaim 1, wherein the polyhydroxylated polyalkenes are present in anamount of from about 5% to about 200% by weight of the therapeuticsubstance.
 23. A formulation according to claim 22, wherein thepolyhydroxylated polyalkene is present between about 10% and about 50%by weight of the substance.
 24. A method for the preparation of aformulation as defined in claim 1, comprising blending the therapeuticagent with the carbohydrate compound or derivative thereof andpolyhydroxylated polyalkene substances in an aqueous vehicle, drying theresulting blend to a powder, and then formulating with propellant.
 25. Amethod according to claim 24, wherein the aqueous vehicle is selectedfrom saline, a suitable buffer, and deionised water.
 26. A methodaccording to claim 24, which comprises spray—drying the blend.
 27. Apowdered formulation of a therapeutic agent, a carbohydrate compound orderivative thereof and a polyhydroxylated polyalkene, as defined inclaims 1, which is suitable for incorporation with a haloalkanepropellant for dispensing from a metered dose inhaler.
 28. A powderedformulation according to claim 27, wherein the powder particles have anaerodynamic diameter of between about 1 μm and 50 μm.
 29. A metered doseinhalation device provided with a reservoir comprising a formulationaccording to claim 1.